Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions

Matthias Kästle,Ardiyanto Liaunardy-Jopeace,Wolfgang W Schamel,John James,Camilla Merten,Roland Hartig,Susana Minguet,Luca Simeoni,Thilo Kaehne,Burkhart Schraven,Nadine M Woessner

doi:10.1186/s12964-020-00673-z

Abstract

BackgroundUpon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood.MethodsKnock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies.ResultsCo-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation.ConclusionsTaken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR.Major findingsOur data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation.FN7ZF7ULbDVsMsT6Sckwh1Video

Highlights

Lck, a member of the Src-family of tyrosine kinases, is primarily expressed in thymocytes and mature T cells [1, 2]
Major findings: Our data change the current view on the function of Y192 and suggest that Y192 regulates Lck activity in a manner independent of Y505 phosphorylation
Kinetics of LckY192 phosphorylation upon T-cell receptor (TCR) stimulation in primary murine T cells In Jurkat T cells, it has previously been shown that LckY192 is constitutively phosphorylated and that TCR stimulation induces a rapid increase in the Y192 phosphorylation [25, 26, 35,36,37]

Summary

Introduction

A member of the Src-family of tyrosine kinases, is primarily expressed in thymocytes and mature T cells [1, 2]. The signaling function of Lck in primary peripheral T cells has been elegantly assessed in transgenic mice in which the expression of Lck can be inducibly regulated [8] These approaches have shown that peripheral Lck−/− T cells are impaired in initiating proximal TCR signaling and display attenuated phosphorylation of TCRζ, ZAP70, LAT, PLC-γ1 as well as altered C a2+ mobilization [9]. These signaling defects correlate with a reduced CD3mediated proliferation and CD69 upregulation [9, 10]. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood

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Results

Discussion

Conclusion